This invention relates to the fields of medical devices, medical diagnostics, and cell counting.
Of the 40.4 million people infected with HIV globally, more than 35 million live in developing countries with significant resource limitations, many of whom are in urgent need of diagnosis, monitoring and antiretroviral therapy. In the process of managing HIV-infected subjects, counts of a specific white blood cell population, CD4+ T lymphocytes, have proven to be essential biological indicators. In adults, the absolute number of CD4+ T cells per microliter of blood has critical prognostic and therapeutic implications and is used for both HIV staging and treatment decisions. Regular monitoring of CD4 counts—two to four times per year—is recommended for all stages of infection. Clinically, a CD4 count below 200 cells mL−1 establishes the diagnosis of AIDS and in most settings is used as a marker to initiate antiretroviral treatment (ART) and prophylaxis against opportunistic infections. Higher CD4 count thresholds of 350 and 500 cells mL−1 are widely used as markers to increase the intensity of monitoring, and in some settings, to initiate ART. However, affordable and appropriate laboratory monitoring tools to determine CD4 counts have little penetration in resource-limited settings, despite ongoing international efforts to extend the availability of ART to these areas.
Currently, the gold standard for CD4+ T cell enumeration is flow cytometric counting of lymphocyte subpopulations using monoclonal antibodies and commercial multi-purpose flow cytometers or single-purpose CD4-counting flow cytometers. Although these instruments are high throughput and accurate, their cost and technical requirements for operation and maintenance have limited their reach and significantly delayed the implementation of HIV treatment programs in resource-limited areas worldwide. Smaller instruments like the Guava EasyCD4 offer limited improvements and have not been widely adopted. Non-cytofluorographic methods, including ELISA and bead format assays have been suggested as useful alternatives for CD4+ T lymphocyte quantification, since these methods require less equipment and have lower reagent costs than flow cytometry. However, they have much lower throughput, are more labor intensive and less accurate, and are not widely used or recommended by World Health Organization guidelines.
In addition, improvements on the back-end aspects of CD4 counting—such as miniaturization of equipment—do not address the most problematic issue for resource-limited settings, which is sample preparation. The requirements to collect blood by venipuncture, to lyse erythrocytes, to centrifuge samples, or to use pipettes for any step in the diagnostic assay are extremely problematic in these settings.
Accordingly there is still a need for low cost methods for the diagnosis and monitoring of CD4 cell populations.